COV scan 3 populations

• This notebook summarizes the results from temporal covariance anaysis from CVTKPY.

1 Results from CVTKPY: genome-wide temporal covariances of allele frequencies

pops<-c("PWS","TB","SS")
covs<-data.frame()
Variance<-data.frame()
winsize<-c("50k","75k","100k","250k")

for (w in 1: length(winsize)){
    covs<-data.frame()
    for (p in 1: length(pops)){
        #covariance output file
        cov<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/3Pops_maf05_temp_cov_matrix_",pops[p],"_",winsize[w],".csv"))
        cov<-cov[,-1]
        
        ci<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/3Pops_maf05_",pops[p],"_Cov_CIs_bootstrap5000_",winsize[w],"window.csv"))
        ci<-ci[,-1]
        
        #reshape the matrix
        mat1<-cov[1:3,]
        mat2<-cov[4:6,]
        
        covdf<-data.frame()
        k=1
        for (i in 1:nrow(mat1)){
            for (j in 1:ncol(mat1)){
                covdf[k,1]<-mat2[i,j]
                covdf[k,2]<-mat1[i,j]
                k=k+1
            }
        }
        colnames(covdf)<-c("label","value")
        covdf$value<-as.numeric(covdf$value)
        covar<-covdf[grep("cov",covdf$label),]
        vars<-covdf[grep("var",covdf$label),]
        
        #remove the redundant values
        if (pops[p]!="SS") covar<-covar[!duplicated(covar[, 2]),] 
        if (pops[p]=="SS") covar<-covar[c(1,2,4),]
        
        #assign the starting time period and covering period values
        covar$year<-c(1,2,2)
        covar$series<-c("1991","1991","1996")
        
        vars$year<-c(1,2,2)
        vars$series<-c("1991","1991","1996")
        
        #assign population name
        covar$location<-pops[p]
        vars$location<-pops[p]
        
        #attach ci info
        covar$ci_l<-c(ci[1,2], ci[1,3],ci[2,3])
        covar$ci_u<-c(ci[4,2], ci[4,3],ci[5,3])
        
        #combine in to one matrix
        covs<-rbind(covs, covar)
        Variance<-rbind(Variance, vars)
    }
    
    covs$ci_l<-as.numeric(covs$ci_l)
    covs$ci_u<-as.numeric(covs$ci_u)
    ggplot(data=covs, aes(x=year, y=value, color=location, shape=series, group=interaction(location, series)))+
        geom_point(size=3, position=position_dodge(width = 0.1,preserve ="total"))+
        #geom_errorbar(data=covs, aes(x=year, y=value, ymin=ci_l, ymax=ci_u), width=.2, size=.2, position=position_dodge(width = 0.1,preserve ="total"))+
        geom_line(data=covs, aes(x=year, y=value,color=location, group=interaction(location, series)), position=position_dodge(width = 0.1,preserve ="total"))+
        ylab("Covariance")+xlab('')+theme_classic()+
        theme(axis.text.x = element_blank(),legend.title = element_blank())+
        geom_hline(yintercept = 0,color="gray70", size=0.3)+
        geom_errorbar(aes(ymin=ci_l, ymax=ci_u), width=.2, size=.2, position=position_dodge(width = 0.1,preserve ="total"))+
        scale_shape_manual(values=c(16,17),labels=c("1991-","1996-"))+
        scale_x_continuous(breaks = c(1,2))
    ggsave(paste0("../Output/COV/3Pops_Cov_overtime_CI_",winsize[w],".window.png"),width = 4.7, height = 3, dpi=300)
}    

2 Find regions with high covariances in each population

• From Temporal Covariance analysis -output covariances for each time period

2.1 Plot the covariances across the genome

#Find the regions with a high temporal covariance 
pops<-c("PWS","TB","SS")
winsize<-"100k"
evens<-paste0("chr",seq(2,26, by=2))
cov.list<-list()
covs_all<-list()
k=1
for (p in 1: length(pops)){
    pop<-pops[p]
    iv<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/3pops_intervals_",winsize,"window.csv"), row.names = 1)
    if (p==3) {
        cov23<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/",pop,"_cov23_2017-2006_2006-1996_3Pops_",winsize,"window.csv"), header = F)
        covs<-cbind(iv, cov23)
        colnames(covs)[4]<-c("cov23")
        covs$index=1:nrow(covs)
        covs$color<-"col1"
        covs$color[covs$chrom %in% evens]<-"col2"

        covs[sapply(covs, is.infinite)] <- NA
        covs[sapply(covs, is.nan)] <- NA
        
        cov.list[[k]]<-covs
        names(cov.list)[k]<-paste0(pop,"_",winsize)    
        k=k+1
            
        y<-min(covs$cov23, na.rm=T)
        ymin<-ifelse (y<=-0.1,-0.1, y) 
        ymax<-max(covs$cov23, na.rm=T)
        ggplot(covs, aes(x=index, y=cov23, color=color))+
            geom_point(size=1, alpha=0.5)+
            theme_classic()+
            ylim(ymin,ymax)+
            scale_color_manual(values=c("gray70","steelblue"), guide="none")+
            ylab("Covariance")+xlab('Chromosome')+
            theme(axis.text.x = element_blank())+
            ggtitle(paste0(pop," ", winsize," window"))
        #ggsave(paste0("../Output/COV/3Pops.",pop,"_tempCovs_acrossGenome_",winsize[i], "Window.png"), width = 8, height = 2.7, dpi=300) 
        }
    else {
        cov12<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/",pop,"_cov12_1996-1991_2006-1996_3Pops_",winsize,"window.csv"), header = F)
        cov23<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/",pop,"_cov23_2017-2006_2006-1996_3Pops_",winsize,"window.csv"), header = F)
        cov13<-read.csv(paste0("~/Projects/Pacherring_Vincent/MD7000/",pop,"_cov13_2017-2006_1996-1991_3Pops_",winsize,"window.csv"), header = F)
        covs<-cbind(iv, cov12, cov23,cov13)
        colnames(covs)[4:6]<-c("cov12","cov23","cov13")
        covs$index=1:nrow(covs)
    
        covs$color<-"col1"
        covs$color[covs$chrom %in% evens]<-"col2"
    
        covs[sapply(covs, is.infinite)] <- NA
        covs[sapply(covs, is.nan)] <- NA
        
        cov.list[[k]]<-covs
        names(cov.list)[k]<-paste0(pop,"_",winsize)    
        k=k+1
        covsm<-melt(covs[,c("index","color","cov12","cov23","cov13")], id.vars = c("index", "color"))
        ymax<-max(covsm$value, na.rm=T)
        y<-min(covsm$value, na.rm=T)
        ymin<-ifelse (y<=-0.1,-0.1, y) 
        ggplot(covsm, aes(x=index, y=value, color=color))+
            facet_wrap(~variable, nrow=3)+
            geom_point(size=1, alpha=0.5)+
            theme_classic()+
            ylim(ymin,ymax)+
            scale_color_manual(values=c("gray70","steelblue"), guide="none")+
            ylab("Covariance")+xlab('Chromosome')+
            theme(axis.text.x = element_blank())+
            ggtitle(paste0(pop," ", winsize," window"))
        #ggsave(paste0("../Output/COV/3Pops.",pop,"_tempCovs_acrossGenome_",winsize, "Window.png"), width = 8, height = 8, dpi=300)    
    }
}

3 Find the covariance lower cut off values

cv<-c("cov12","cov13","cov23")
cvrange<-data.frame(pop=c(paste0(pops[1:2],"_", cv[1]),paste0(pops[1:2],"_", cv[2]),paste0(pops,"_", cv[3])))
k=1
for (i in 1:length(cv)){
    if (i==1|i==2){
        if (i==1) k=1
        if (i==2) k=3
        #PWS
        df1<-cov.list[[paste0("PWS_100k")]]
        df1<-df1[order(df1[,cv[i]], decreasing=T),]
        n<-ceiling(nrow(df1)*0.01) #top1% region
        df1$top1<-"N"
        df1$top1[1:n]<-"PWS"
        rg<-range(df1[df1$top1=="PWS",cv[i]], na.rm=T)
        cvrange[k,"100k"]<-paste0(rg[1],"-",rg[2])
          
        #tb
        df2<-cov.list[["TB_100k"]]
        df2<-df2[order(df2[,cv[i]], decreasing=T),]
        df2$top1<-"N"
        df2$top1[1:n]<-"TB"
        rg2<-range(df2[df2$top1=="TB", cv[i]], na.rm=T)
        cvrange[(k+1),"100k"]<-paste0(rg2[1],"-",rg2[2])
    }
   
    if (i==3){
        k=5
        #pws
        df1<-cov.list[["PWS_100k"]]
        df1<-df1[,c("chrom","start","end","cov23")]
        df1<-df1[order(df1$cov23, decreasing=T),]
        n<-ceiling(nrow(df1)*0.01) #top1% region
        df1$top1<-"N"
        df1$top1[1:n]<-"PWS"
        
        rg<-range(df1[df1$top1=="PWS",cv[i]], na.rm=T)
        cvrange[k,"100k"]<-paste0(rg[1],"-",rg[2])
           
        #tb
        df2<-cov.list[["TB_100k"]]
        df2<-df2[,c("chrom","start","end","cov23")]
        df2<-df2[order(df2$cov23, decreasing=T),]
        df2$top1<-"N"
        df2$top1[1:n]<-"TB"
        rg2<-range(df2[df2$top1=="TB", cv[i]], na.rm=T)
        cvrange[(k+1),"100k"]<-paste0(rg2[1],"-",rg2[2])
    
        #ss
        df3<-cov.list[["SS_100k"]]
        df3<-df3[,c("chrom","start","end","cov23")]
        df3<-df3[order(df3$cov23, decreasing=T),]
        df3$top1<-"N"
        df3$top1[1:n]<-"SS"
        rg3<-range(df3[df3$top1=="SS", cv[i]], na.rm=T)
        cvrange[(k+2),"100k"]<-paste0(rg3[1],"-",rg3[2])
        }
    }
}

cvs<-melt(cvrange, id.vars = "pop")
cvs<-cvs %>%
  separate(value, c("low", "high"), "-")
cvs$low<-as.numeric(cvs$low)
cvs$high<-as.numeric(cvs$high)
cvs<-cvs%>%
  separate(pop, c("pop", "cov"), "_")

ggplot(cvs, aes(x=cov, y=high, fill=pop))+
    geom_crossbar(aes(ymin=low, ymax=high), width=0.5, position=position_dodge(width = 1))+
    ylab("Range of covariances")+
    theme_light()+xlab("")+
    geom_vline(xintercept=c(1.5,2.5), color="gray")+
    theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank(), legend.title=element_blank())+
    ggtitle("Top1% Cov Range")
ggsave("../Output/COV/COVscan_3pop/TempCov_Range_comparison_100k.png", width = 5, height = 3, dpi=300)

ggplot(cvs, aes(x=cov, y=low, color=pop))+
    geom_point()+
    ylab("Lower limit of top 1% covariance")+
    theme_light()+xlab("")+
    geom_vline(xintercept=c(1.5,2.5), color="gray")+
    theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank(), legend.title=element_blank())
ggsave("../Output/COV/COVscan_3pop/TempCov_Range_lowLimit_comparison_100k.png", width = 5, height = 3, dpi=300)

3.1 Use the PWS’s lowest covariance values to definte outlier regions for all populations

lows<-cvs[cvs$pop=="PWS",]
names(lows)[3]<-"window"
#low for PWS 100k
#   pop   cov variable        low      high
#15 PWS cov12     100k 0.02874841 0.0821782
#17 PWS cov13     100k 0.03102712 0.1036491
#19 PWS cov23     100k 0.03274974 0.2022322


# Outliers based on the new low cut-off values 100k window. 
cov12<-data.frame()
cov23<-data.frame()
cov13<-data.frame()

for (i in 1:length(cov.list)){
 #PWS and TB
  if (i==1|i==2){
    covs<-cov.list[[i]]
    pop<-gsub("_.+",'', names(cov.list)[i])
  
    #outlier cutoff value
    x<-lows$low[lows$cov=="cov12"]
    covs12_top<-subset(covs, cov12>=x)
    covs12_top<-covs12_top[order(covs12_top$chrom, covs12_top$start),]
    covs12_top$window<-"100k"
    covs12_top$pop<-pop
    cov12<-rbind(cov12, covs12_top)
    
    covs<-covs[order(covs$cov13, decreasing=T),]
    x<-lows$low[lows$window==win&lows$cov=="cov13"]
    covs13_top<-subset(covs, cov13>=x)
    covs13_top<-covs13_top[order(covs13_top$chrom, covs13_top$start),]
    covs13_top$window<-win
    covs13_top$pop<-pop
    cov13<-rbind(cov13, covs13_top)
    
    covs<-covs[order(covs$cov23, decreasing=T),]
    x<-lows$low[lows$window==win&lows$cov=="cov23"]
    covs23_top<-subset(covs[,c("chrom","start","end","cov23","index","color")], cov23>=x)
    covs23_top<-covs23_top[order(covs23_top$chrom, covs23_top$start),]
    covs23_top$window<-win
    covs23_top$pop<-pop
    cov23<-rbind(cov23, covs23_top)
 }
 if (grepl("SS",names(cov.list)[i])){
    covs<-cov.list[[i]]
    
    pop<-gsub("_.+",'', names(cov.list)[i])
    win<-gsub(paste0(pop,"_"), '', names(cov.list)[i])
    
    covs<-covs[order(covs$cov23, decreasing=T),]
    x<-lows$low[lows$window==win&lows$cov=="cov23"]
    covs23_top<-subset(covs, cov23>=x)
    covs23_top<-covs23_top[order(covs23_top$chrom, covs23_top$start),]
    covs23_top$window<-win
    covs23_top$pop<-pop
    cov23<-rbind(cov23, covs23_top)
    }
}

write.csv(cov12, "../Output/COV/COVscan_3pop/cutoff/3pops_top1percent_outlier_cutoffPWS.cov12.csv",row.names = F)
write.csv(cov23, "../Output/COV/COVscan_3pop/cutoff/3pops_top1percent_outlier_cutoffPWS.cov23.csv",row.names = F)
write.csv(cov13, "../Output/COV/COVscan_3pop/cutoff/3pops_top1percent_outlier_cutoffPWS.cov13.csv",row.names = F)

3.2 Create plots with different colors for outliers

#for COV12 and COV13 for TB and PWS (100K)
cv<-c("cov12","cov13","cov23")
winsize<-"100k"

for (i in 1:length(cv)){
    if (i==1|i==2){
        #cutoff value
        x<-lows$low[lows$cov==cv[i]]
        #PWS
        df1<-cov.list[["PWS_100k"]]
        df1<-df1[order(df1[,cv[i]], decreasing=T),]
        df1$top1<-"N"
        df1$top1[df1[,cv[i]]>=x]<-"PWS"
        
        #tb
        df2<-cov.list[["TB_100k"]]
        df2<-df2[order(df2[,cv[i]], decreasing=T),]
        df2$top1<-"N"
        df2$top1[df2[,cv[i]]>=x]<-"TB"
        
        #Combine PWS and TB tables
        co<-rbind(df1, df2)
        co$chrom<-factor(co$chrom, levels=paste0("chr", 1:26))
        co$top1<-factor(co$top1, levels=c("PWS","TB","N"))
        colnames(co)[which(colnames(co)==cv[i])]<-"cov"
    
        ymax<-max(co$cov, na.rm=T)
        #Plot each genome separately
        ggplot(co, aes(x=start/1000000, y=cov, color=top1))+
            geom_point(size=0.5, alpha=0.6)+
            facet_wrap(~chrom, ncol=4)+
            theme_classic()+ylim(-0.1,ymax)+
            scale_color_manual(values=c("deeppink","blue" ,"gray80"), labels=c("PWS", "TB", ""))+
            ylab("Covariance")+xlab('Postion (Mb)')+
            ggtitle(paste0(winsize[w]," window ",cv[i]))+
            scale_x_continuous(labels = comma)+
            guides(color = guide_legend(override.aes = list(color=c("deeppink","blue","white")), title=element_text("Top 1%")))
   
        ggsave(paste0("../Output/COV/COVscan_3pop/cutoff/3Pops.",cv[i],"_perChrom_",winsize, "Window_Outliers.png"), width = 10, height = 8, dpi=300)
        
        #Whole geonome in 1 plot 
        #count the number of sites per chromosomes
              #assgin colors
        co$top1<-apply(co, 1, function(x) {ifelse (x['top1']=="N", x['color'], x['top1'])} )
        co$top1<-factor(co$top1, levels=c("PWS","TB","col1","col2"))
        
        poss<-data.frame(chr=paste0("chr",1:26))
        k=1
        for (j in 1:26){
            df<-df1[df1$chr==paste0("chr",j),]
            poss$start[j]<-k
            poss$end[j]<-k+nrow(df)-1
            k=k+nrow(df)
        }
        poss$x<-poss$start+(poss$end-poss$start)/2
        ymax<-max(co$cov, na.rm=T)
        ggplot(co, aes(x=index, y=cov, color=top1))+
            geom_point(size=0.5)+
            theme_classic()+ylim(-0.1,ymax)+
            scale_color_manual(values=c("#FF3293B3","#0433FFB3" ,"#ADD8E680","#C0C0C088"), labels=c("PWS", "TB", "",""))+
                ylab("Covariance")+
                ggtitle(paste0(" 100k window ",cv[i]))+
                guides(color = guide_legend(override.aes = list(color=c("deeppink","#0433FF","white","white"), size=2), title=element_text("Outlier Region", size=10)))+
            scale_x_continuous(name="Chromosome", breaks=poss$x, labels=1:26)
        ggsave(paste0("../Output/COV/COVscan_3pop/cutoff/3Pops.",cv[i],"_100k_Window_Outliers.png"), width = 10, height = 3.5, dpi=300)
    }
   
    if (i==3){
       #cutoff value
        x<-lows$low[lows$cov==cv[i]]
        #PWS
        df1<-cov.list[["PWS_100k"]]
        df1<-df1[,c("chrom","start","end","cov23","index","color")]
        df1<-df1[order(df1$cov23, decreasing=T),]
        df1$top1<-"N"
        df1$top1[df1[,cv[i]]>=x]<-"PWS"
        
        #tb
        df2<-cov.list[["TB_100k"]]
        df2<-df2[,c("chrom","start","end","cov23","index","color")]
        df2<-df2[order(df2$cov23, decreasing=T),]
        df2$top1<-"N"
        df2$top1[df2[,cv[i]]>=x]<-"TB"
    
        #ss
        df3<-cov.list[["SS_100k"]]
        df3<-df3[,c("chrom","start","end","cov23","index","color")]
        df3<-df3[order(df3$cov23, decreasing=T),]
        df3$top1<-"N"
        df3$top1[df3[,cv[i]]>=x]<-"SS"

        co<-rbind(df1,df2,df3)

        co$chrom<-factor(co$chrom, levels=paste0("chr", 1:26))
        co$top1<-factor(co$top1, levels=c("PWS","TB","SS","N"))
        ymax<-max(co$cov23, na.rm=T)
        ggplot(co, aes(x=start/1000000, y=cov23, color=top1))+
            geom_point(size=0.5, alpha=0.6)+
            facet_wrap(~chrom, ncol=4)+
            theme_classic()+ylim(-0.1,ymax)+
            ylab("Covariance")+xlab('Postion (Mb)')+
            ggtitle(paste0(winsize[w]," window ",cv[i]))+
            scale_x_continuous(labels = comma)+
            #scale_color_discrete(breaks=c("PWS","SS","TB"))+
            scale_color_manual(values=c("deeppink","blue",gre,"gray80"), labels=c("PWS","TB","SS", ""))+
            guides(color = guide_legend(override.aes = list(color=c("deeppink","blue",gre,         "white")),title=element_text("Top 1% outliers"))) 
        ggsave(paste0("../Output/COV/COVscan_3pop/cutoff/3Pops.cov23_3Pops_perChrom_",winsize, "Window_Outliers.png"), width = 10, height = 9, dpi=300)
        
        #assgin colors
        co$top1<-apply(co, 1, function(x) {ifelse (x['top1']=="N", x['color'], x['top1'])} )
        co$top1<-factor(co$top1, levels=c("PWS","TB","SS","col1","col2"))
        #count the number of sites per chromosomes
        poss<-data.frame(chr=paste0("chr",1:26))
        k=1
        for (j in 1:26){
            df<-df1[df1$chr==paste0("chr",j),]
            poss$start[j]<-k
            poss$end[j]<-k+nrow(df)-1
            k=k+nrow(df)
        }
        poss$x<-poss$start+(poss$end-poss$start)/2
        ymax<-max(co$cov, na.rm=T)
        ggplot(co, aes(x=index, y=cov23, color=top1))+
            geom_point(size=0.5, alpha=0.6)+
            theme_classic()+ylim(-0.1,ymax)+
            scale_color_manual(values=c("#FF3293B3","#0433FFB3","#008F00B3" ,"#ADD8E680","#C0C0C088"), labels=c("PWS", "TB","SS", "",""))+
                ylab("Covariance")+
                ggtitle(paste0(" 100k window ",cv[i]))+
                guides(color = guide_legend(override.aes = list(color=c("deeppink","#0433FF","#008F00","white","white"), size=2), title=element_text("Outlier (1%)")))+
            scale_x_continuous(name="Chromosome", breaks=poss$x, labels=1:26)+
            theme(legend.title = element_text(size=10))
        ggsave(paste0("../Output/COV/COVscan_3pop/cutoff/3Pops.",cv[i],"_100k_Window_Outliers.png"), width = 10, height = 3.5, dpi=300)
        }
        
        }

3.2.1 Whole genome plots all time priods for PWS and TB

## Plot 3 time periods together for PWS and TB
Cov<-data.frame()
for (i in 1:length(cv)){
    #cutoff value
    x<-lows$low[lows$cov==cv[i]]
    #PWS
    df1<-cov.list[["PWS_100k"]]
    df1<-df1[order(df1[,cv[i]], decreasing=T),]
    df1$top1<-"N"
    df1$top1[df1[,cv[i]]>=x]<-"PWS"
    
    #tb
    df2<-cov.list[["TB_100k"]]
    df2<-df2[order(df2[,cv[i]], decreasing=T),]
    df2$top1<-"N"
    df2$top1[df2[,cv[i]]>=x]<-"TB"
    
    #Combine PWS and TB tables
    co<-rbind(df1, df2)
    co$chrom<-factor(co$chrom, levels=paste0("chr", 1:26))
    colnames(co)[which(colnames(co)==cv[i])]<-"cov"
    #assgin colors
    co$top1<-apply(co, 1, function(x) {ifelse (x['top1']=="N", x['color'], x['top1'])} )
    co$top1<-factor(co$top1, levels=c("PWS","TB","col1","col2"))
    co$time<-cv[i]
    
    Cov<-rbind(Cov, co[,c("index", "cov","top1","time")])
}

#count the number of sites per chromosomes
df1<-cov.list[["PWS_100k"]]
poss<-data.frame(chr=paste0("chr",1:26))
k=1
for (j in 1:26){
        df<-df1[df1$chr==paste0("chr",j),]
        poss$start[j]<-k
        poss$end[j]<-k+nrow(df)-1
        k=k+nrow(df)
}
poss$x<-poss$start+(poss$end-poss$start)/2
ymax<-max(co$cov, na.rm=T)
ggplot(Cov, aes(x=index, y=cov, color=top1))+
    facet_wrap(~time, ncol=1)+
    geom_point(size=0.5)+
    theme_classic()+ylim(-0.1,ymax)+
    scale_color_manual(values=c("#FF3293B3","#0433FFB3" ,"#ADD8E680","#C0C0C088"), labels=c("PWS", "TB", "",""))+
    ylab("Covariance")+
    guides(color = guide_legend(override.aes = list(color=c("deeppink","#0433FF","white","white"), size=2), title=element_text("Outlier", size=10)))+
    scale_x_continuous(name="Chromosome", breaks=poss$x, labels=1:26)

ggsave(paste0("../Output/COV/COVscan_3pop/cutoff/PWS_TB_100k_Window_Outliers.png"), width = 11, height = 5, dpi=300)
        }}

3.3 Overlapping outlier regions between different populations

#100k
cv<-c("cov12","cov13","cov23")
pairs<-t(combn(pops, 2))
pairs<-data.frame(pairs)
colnames(pairs)<-paste0("pop",1:2)
Ov_direct<-data.frame(cov=c(cv[1:2],"cov23-PT","cov23-PS","cov23-ST" ,"cov23-3"))
Ov_300<-data.frame(cov=c(cv[1:2],"cov23-PT","cov23-PS","cov23-ST" ,"cov23-3"))
for (i in 1:length(cv)){
    df<-read.csv(paste0("../Output/COV/COVscan_3pop/cutoff/3pops_top1percent_outlier_cutoffPWS.", cv[i], ".csv"))
    df$id<-paste0(df$chrom,"_",df$start)
    
    if (i!=3){
        #exact overlaps
        isec<-intersect(df$id[df$pop=="PWS"], df$id[df$pop=="TB"]) 
        Ov_direct$count[i]<-length(isec)
        
        #### Check chromosome region overlap +-200,000 bases
        pop1<-df[df$pop=="PWS",]
        pop2<-df[df$pop=="TB",]
        overlps<-data.frame()
        overlps2<-data.frame()
        for (n in 1: nrow(pop1)){
            re<-pop2[pop2$chrom==pop1$chrom[n],]
            if (nrow(re)>=1){
                for (s in 1: nrow(re)){
                    if (re$start[s]<=pop1$start[n]+200000 & re$start[s]>=pop1$start[n]-200000){
                        overlps<-rbind(overlps, re[s,])
                        overlps2<-rbind(overlps2,pop1[n,])}
                }
            }
        }
        # Merge two tables into one summary overlap table:
        ov<-data.frame(id=overlps$id)
        for (n in 1: nrow(overlps)){
            if (overlps$start[n]<overlps2$start[n]) {ov$start[n]<-overlps$start[n]; ov$end[n]<-overlps2$end[n]}
            if (overlps$start[n]>=overlps2$start[n]) {ov$start[n]<-overlps2$start[n];ov$end[n]<-overlps$end[n]}
        }
        ov[,"cov.PWS"]<-overlps[,4]
        ov[,"cov.TB"]<-overlps2[,4]
        write.csv(ov, paste0("../Output/COV/COVscan_3pop/cutoff/Overlap_regions_",cv[i],"_plusminus100k.csv"), row.names = F)
        Ov_300$count[i]<-nrow(ov)
        }
        
    if (i==3){
        isec<-intersect(df$id[df$pop=="PWS"], df$id[df$pop=="TB"]) 
        isec2<-intersect(df$id[df$pop=="PWS"], df$id[df$pop=="SS"]) 
        isec3<-intersect(df$id[df$pop=="SS"], df$id[df$pop=="TB"]) 
        Ov_direct$count[i]<-length(isec)
        Ov_direct$count[i+1]<-length(isec2)
        Ov_direct$count[i+2]<-length(isec3)
        Ov_direct$count[i+3]<-length(intersect(df$id[df$pop=="SS"], intersect(df$id[df$pop=="PWS"], df$id[df$pop=="TB"])))
        
        for(j in 1:nrow(pairs)){
        #### Check chromosome region overlap +-200,000 bases
            pop1<-df[df$pop==pairs[j,1],]
            pop2<-df[df$pop==pairs[j,2],]
            overlps<-data.frame()
            overlps2<-data.frame()
            for (n in 1: nrow(pop1)){
                re<-pop2[pop2$chrom==pop1$chrom[n],]
                if (nrow(re)>=1){
                    for (s in 1: nrow(re)){
                        if (re$start[s]<=pop1$start[n]+200000 & re$start[s]>=pop1$start[n]-200000){
                            overlps<-rbind(overlps, re[s,])
                            overlps2<-rbind(overlps2,pop1[n,])}
                    }
                }
            }
        # Merge two tables into one summary overlap table:
            ov<-data.frame(id=overlps$id)
            for (n in 1: nrow(overlps)){
                if (overlps$start[n]<overlps2$start[n]) {ov$start[n]<-overlps$start[n]; ov$end[n]<-overlps2$end[n]}
                if (overlps$start[n]>=overlps2$start[n]) {ov$start[n]<-overlps2$start[n];ov$end[n]<-overlps$end[n]}
            }
        
            ov[,paste0("cov.",pairs[j,1])]<-overlps[,4]
            ov[,paste0("cov.",pairs[j,2])]<-overlps2[,4]
            ov<-ov[!duplicated(ov),]
            write.csv(ov, paste0("../Output/COV/COVscan_3pop/cutoff/Overlap_regions_",cv[i],"_",pairs[j,1],".", pairs[j,2],"_plusminus200k.csv"), row.names = F)
            Ov_300$count[i+j-1]<-nrow(ov)
    }
    }
}
write.csv(Ov_direct, paste0("../Output/COV/COVscan_3pop/cutoff/Direct_Overlapping_regions_counts_3pop_summary.csv"))
Ov_300$count[6]<-NA
write.csv(Ov_300, paste0("../Output/COV/COVscan_3pop/cutoff/Overlapping_regions_counts_3pop_plusMinus200k.csv"))

4 Run the snpEff pipeline to find annotation in the outlier regions (100k-window+-100k)

4.1 Create a script to run SnpEff

Create VCF files with selected regions & run snpEff

#Create bed files
cv<-c("cov12","cov13","cov23")
#Prevent scientific notation in bed files
options(scipen=999)

#The first line of bed files is often not red by vcftools
for (i in 1:3){
    df<-read.csv(paste0("../Output/COV/COVscan_3pop/cutoff/3pops_top1percent_outlier_cutoffPWS.", cv[i], ".csv"))
    #add 100k
    df$start<-df$start-100000
    df$end<-df$end+100000
    dfp<-df[df$pop=="PWS",1:3]
    colnames(dfp)<-c('track type=bedGraph', '1','1')
    write.table(dfp, paste0("../Output/COV/COVscan_3pop/cutoff/PWS_outliers_",cv[i],"_new.bed"),quote = F, row.names = F, col.names = T,sep = "\t")
    dft<-df[df$pop=="TB",1:3]
    colnames(dft)<-c('track type=bedGraph', '1','1')
    write.table(dft, paste0("../Output/COV/COVscan_3pop/cutoff/TB_outliers_",cv[i],"_new.bed"),quote = F, row.names = F, col.names = F,sep = "\t")
    
    if (i==3){
        dfs<-df[df$pop=="SS",1:3]
        colnames(dfs)<-c('track type=bedGraph', '1','1')
        write.table(dfs, paste0("../Output/COV/COVscan_3pop/cutoff/SS_outliers_",cv[i],"_new.bed"),quote = F, row.names = F, col.names = F,sep = "\t")
    }
}

# Create a bash script to create vcf files with selected regions
bedfiles<-list.files("../Output/COV/COVscan_3pop/cutoff/", pattern="*_new.bed")

sink("../COVscan_createVCFs_3Pops_cutoff.sh")
cat("#!/bin/bash \n\n")
for (i in 1:length(bedfiles)){
    fname<-gsub(".bed",'', bedfiles[i])
    cat(paste0("vcftools --gzvcf Data/new_vcf/3pop/3pops.MD7000_NS0.5_maf05.vcf.gz --bed Output/COV/COVscan_3pop/cutoff/", bedfiles[i], " --out Output/COV/COVscan_3pop/cutoff/", fname," --recode --keep-INFO-all \n"))
}
sink(NULL)  

#create a bash script to run snpEff
vfiles<-list.files("../Output/COV/COVscan_3pop/cutoff/", pattern=".recode.vcf")

sink("~/programs/snpEff/runsnpEff_cov_3pop_cutoff.sh")
cat("#!/bin/bash \n\n")
for (i in 1:length(vfiles)){
    fname<-gsub("_new.recode.vcf","",vfiles[i])
    cat(paste0("java -Xmx8g -jar snpEff.jar Ch_v2.0.2.99 ~/Projects/PacHerring/Output/COV/COVscan_3pop/cutoff/",vfiles[i], " -stats ~/Projects/PacHerring/Output/COV/COVscan_3pop/cutoff/",fname,".html >  ~/Projects/PacHerring/Output/COV/COVscan_3pop/cutoff/Anno.",fname,".vcf \n"))
    
    #extract the annotation information
    cat(paste0("bcftools query -f '%CHROM %POS %INFO/AF %INFO/ANN\\n' ~/Projects/PacHerring/Output/COV/COVscan_3pop/cutoff/Anno.",fname,".vcf > ~/Projects/PacHerring/Output/COV/COVscan_3pop/cutoff/",fname,"_annotation \n\n"))

}
sink(NULL)  

4.2 Create summary gene files from snpEff and check overlapping genes.

## Create summary files of snpEff results (gene annotations in the regions of interest) and reformat as a ShinyGo input 

#create gene list 
gfiles<-list.files("../Output/COV/COVscan_3pop/", pattern="genes.txt")

for (i in 1:length(gfiles)){
    df<-read.table(paste0("../Output/COV/COVscan_3pop/",gfiles[i]), sep="\t")
    df<-df[,1:7]
    colnames(df)<-c("GeneName","GeneId","TranscriptId","BioType","variants_impact_HIGH","variants_impact_LOW",  "variants_impact_MODERATE")
    
    fname<-gsub(".genes.txt","",gfiles[i])
    genes<-unique(df$GeneId)
    sink(paste0("../Output/COV/COVscan_3pop/geneIDlist_",fname,".txt"))
    cat(paste0(genes,"; "))
    sink(NULL)
}

#Annotation infor from SnpEff
cv<-c("cov12","cov13","cov23")
for (c in 1:3){
    if (c!=3){
    for (p in 1:2){
        ano<-read.table(paste0("../Output/COV/COVscan_3pop/",pops[p],"_outliers_",cv[c],"_annotation"), header = F)
        annotations<-data.frame()
        for (i in 1: nrow(ano)){
            anns<-unlist(strsplit(ano$V4[i], "\\,|\\|"))
            annm<-data.frame(matrix(anns,ncol = 16, byrow = TRUE))
            annm<-annm[,c(2,3,4,5,8)]
            colnames(annm)<-c("Effect","Putative_impact","Gene_name","Gene_ID","Feature type")
            annm<-annm[!duplicated(annm), ]
            annm$chr<-ano$V1[i]
            annm$pos<-ano$V2[i]
            annm$AF<- ano$V3[i]
            annotations<-rbind(annotations, annm)
        }     
        annotations<-annotations[,c(6:8,1:5)]
        annotations<-annotations[!duplicated(annotations[,1:2]),]
        write.csv(annotations, paste0("../Output/COV/COVscan_3pop/Genes_",pops[p],"_outliers_100k_",cv[c],".csv"), row.names = F)
    }
    }
    if (c==3){
        for (p in 1:3){
        ano<-read.table(paste0("../Output/COV/COVscan_3pop/",pops[p],"_outliers_",cv[c],"_annotation"), header = F)
        annotations<-data.frame()
        for (i in 1: nrow(ano)){
            anns<-unlist(strsplit(ano$V4[i], "\\,|\\|"))
            annm<-data.frame(matrix(anns,ncol = 16, byrow = TRUE))
            annm<-annm[,c(2,3,4,5,8)]
            colnames(annm)<-c("Effect","Putative_impact","Gene_name","Gene_ID","Feature type")
            annm<-annm[!duplicated(annm), ]
            annm$chr<-ano$V1[i]
            annm$chr<-ano$V1[i]
            annm$pos<-ano$V2[i]
            annm$AF<- ano$V3[i]
            annotations<-rbind(annotations, annm)
        }     
        annotations<-annotations[,c(6:8,1:5)]
        annotations<-annotations[!duplicated(annotations[,1:2]),]
        write.csv(annotations, paste0("../Output/COV/COVscan_3pop/Genes_",pops[p],"_outliers_100k_",cv[c],".csv"), row.names = F)
    }
}
  
}

4.3 Find the overlapping gene names

gnamesfiles<-list.files("../Output/COV/COVscan_3pop/", pattern="Genes_.+outliers_100k.+\\d.csv$")

for (i in 1:length(gnamesfiles)){
    df<-read.csv(paste0("../Output/COV/COVscan_3pop/",gnamesfiles[i]))
    df<-df[,c(1,6:7)]
    df<-df[!duplicated(df),]
    
    fname<-gsub(".csv","", gnamesfiles[i])
    fname<-gsub("Genes_","", fname)
    
    
    #add gene names for front and back of intergenic regions
    df2<-df[grep("-", df$Gene_ID),]
    k=1
    df_div<-data.frame()
    oddnames<-data.frame()
    for (j in 1:nrow(df2)){
        names<-unlist(strsplit(df2$Gene_name[j], "-"))
        ids<-unlist(strsplit(df2$Gene_ID[j], "-"))
        
        if (length(names)==2){
            df_div<-rbind(df_div, c(df2$chr[j],names[1],ids[1]))
            k=k+1
            df_div<-rbind(df_div, c(df2$chr[j],names[2],ids[2]))
            k=k+1
        }
       
        if (length(names)!=2){
            n<-grep("si:", names)
            if (length(n)>0){
                if (n==1) newnames<-c(paste0(names[1],"-",names[2]), names[3])
                if (n==2) newnames<-c(names[1],paste0(names[2],"-",names[3]))
                df_div<-rbind(df_div, c(df2$chr[j],newnames[1],ids[1]))
                k=k+1
                df_div<-rbind(df_div, c(df2$chr[j],newnames[2],ids[2]))
                k=k+1
            }
            
            if (length(n)==0) {
                oddnames<-rbind(oddnames, df2[j,])
            }
        }
    }
    df_div<-df_div[!duplicated(df_div),]
    df_div<-df_div[df_div$Gene_ID!="CHR_END",]
    df_div<-df_div[df_div$Gene_ID!="CHR_START",]
    
    remove<-grep("-", df$Gene_ID)
    df<-df[-remove,]
    df<-rbind(df, df_div)
    df<-df[!duplicated(df),]
    
    if (nrow(oddnames)!=0){
        write.csv(df, paste0("../Output/COV/COVscan_3pop/",fname,"GeneList_withIntergenicGenes.csv" ), row.names = F)
        write.csv(oddnames, paste0("../Output/COV/COVscan_3pop/Oddnames_", fname,".csv"))
    }
    if (nrow(oddnames)==0){
        write.csv(df, paste0("../Output/COV/COVscan_3pop/",fname,"GeneList_withIntergenicGenes_new.csv" ), row.names = F)
     }
}
   

## !! ##
## Manually change the oddnames and add them eteo the GeneList files 
#(updated file names has "_new" at the end)

#aggregate all gene names
gnew<-list.files("../Output/COV/COVscan_3pop/", pattern="GeneList_withIntergenicGenes_new.csv$")
Genes<-data.frame()
GeneList<-list()
for (i in 1:length(gnew)){
    df<-read.csv(paste0("../Output/COV/COVscan_3pop/", gnew[i]))
    GeneList[[i]]<-df
    fname<-gsub("GeneList_withIntergenicGenes_new.csv",'',gnew[i])
    names(GeneList)[i]<-fname
    dup<-df[duplicated(df),]
    df<-df[!duplicated(df),]
    Genes<-rbind(Genes, df)
    Genes<-Genes[!duplicated(Genes),]
    
}


#1. Between populations
times<-c("cov12","cov13","cov23")
common<-list()
common_summary<-data.frame(time=times)
for (i in 1:3){
    tlist<-GeneList[grep(times[i], names(GeneList))]
    if (i !=3){
        common_genes<-intersect(tlist[[1]]["Gene_name"], tlist[[2]]["Gene_name"])
        common[[i]]<-common_genes
        names(common)[[i]]<-times[i]
        common_summary$PWS[i]<-nrow(tlist[[grep("PWS", names(tlist))]])
        common_summary$TB[i]<-nrow(tlist[[grep("TB", names(tlist))]])
        common_summary$SS[i]<-NA
        common_summary$common_PWS.TB[i]<-nrow(common_genes)
        
        pws<-tlist[[1]]["Gene_name"]
        tb<-tlist[[2]]["Gene_name"]
        x<-list(PWS=pws$Gene_name,TB=tb$Gene_name)
        ggvenn(x, fill_color = cols[c(2,1)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle(times[i])
        ggsave(paste0("../Output/COV/COVscan_3pop/Venn_",times[i],".png"), width = 3, height=3, dpi=300)
    }
    if (i==3){
        common_summary$PWS[i]<-nrow(tlist[[grep("PWS", names(tlist))]])
        common_summary$TB[i]<- nrow(tlist[[grep("TB", names(tlist))]])
        common_summary$SS[i]<- nrow(tlist[[grep("SS", names(tlist))]])
        
        genes1<-intersect(tlist[[1]]["Gene_name"], tlist[[3]]["Gene_name"])
        genes2<-intersect(tlist[[1]]["Gene_name"], tlist[[2]]["Gene_name"])
        genes3<-intersect(tlist[[2]]["Gene_name"], tlist[[3]]["Gene_name"])
        genes4<-intersect(tlist[[1]]["Gene_name"],intersect(tlist[[2]]["Gene_name"], tlist[[3]]["Gene_name"]))
        common_summary$common_PWS.TB[i]<-nrow(genes1)
        common_summary$common_PWS.SS[i]<-nrow(genes2)
        common_summary$common_SS.TB[i]<-nrow(genes3)
        common_summary$common3[i]<-nrow(genes4)
        k=i
        common[[k]]<-genes2
        names(common)[[k]]<-paste0(times[i],"_PWS.SS")
        k=k+1
        common[[k]]<-genes1
        names(common)[[k]]<-paste0(times[i],"_PWS.TB")
        k=k+1
        common[[k]]<-genes3
        names(common)[[k]]<-paste0(times[i],"_SS.TB")
        k=k+1
        common[[k]]<-genes4
        names(common)[[k]]<-paste0(times[i],"_3pops")
        
        pws<-tlist[[1]]["Gene_name"]
        tb<-tlist[[3]]["Gene_name"]
        ss<-tlist[[2]]["Gene_name"]
        x<-list(PWS=pws$Gene_name,TB=tb$Gene_name, SS=ss$Gene_name)
        ggvenn(x, fill_color = cols[c(2,1,3)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle(times[i])
        ggsave(paste0("../Output/COV/COVscan_3pop/Venn_",times[i],".png"), width = 4, height=4, dpi=300)
        
         x1<-list(PWS=pws$Gene_name,TB=tb$Gene_name)
        ggvenn(x1, fill_color = cols[c(2,1)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle(times[i])
        ggsave(paste0("../Output/COV/COVscan_3pop/Venn_PWS_TB_",times[i],".png"), width = 3, height=3, dpi=300)
         x2<-list(PWS=pws$Gene_name,SS=ss$Gene_name)
        ggvenn(x2, fill_color = cols[c(2,3)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle(times[i])
        ggsave(paste0("../Output/COV/COVscan_3pop/Venn_PWS_SS_",times[i],".png"), width = 3, height=3, dpi=300)
          x3<-list(SS=ss$Gene_name, TB=tb$Gene_name)
        ggvenn(x3, fill_color = cols[c(3,1)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle(times[i])
        ggsave(paste0("../Output/COV/COVscan_3pop/Venn_SS_TB_",times[i],".png"), width = 3, height=3, dpi=300)
        
        
        }
}
write.csv(common_summary, "../Output/COV/COVscan_3pop/Common_genes_withIntergenes_3pops.csv")


#What are the overlapping gene names between populations
common_times<-list()
for (i in 1: length(common)){
    gids<-common[[i]]
    df<-data.frame(Gene_name=gids)
    
    df2<-merge(df, Genes, by="Gene_name")
    write.csv(df2, paste0("../Output/COV/COVscan_3pop/Common_genes_", names(common)[i],".csv"), row.names = F)
    common_times[[i]]<-df2
    names(common_times)[i]<- names(common)[i]
}

#non_overlapping genes COV23

tlist<-GeneList[grep(times[3], names(GeneList))]
pws23<-tlist[[1]]["Gene_ID"]
ss23<-tlist[[2]]["Gene_ID"]
tb23<-tlist[[3]]["Gene_ID"]
genes1<-intersect(tlist[[1]]["Gene_ID"], tlist[[3]]["Gene_ID"])
genes2<-intersect(tlist[[1]]["Gene_ID"], tlist[[2]]["Gene_ID"])
genes3<-intersect(tlist[[2]]["Gene_ID"], tlist[[3]]["Gene_ID"])
genes4<-intersect(tlist[[1]]["Gene_ID"],intersect(tlist[[2]]["Gene_ID"], tlist[[3]]["Gene_ID"]))

overlaps<-rbind(genes1, genes2, genes3, genes4)
overlaps<-overlaps[!duplicated(overlaps$Gene_ID),]
pws23_only<-data.frame(Gene_ID=pws23[!(pws23$Gene_ID %in% overlaps), ])
g1<-unique(pws23_only$Gene_ID)
sink("../Output/COV/COVscan_3pop/genes/GeneID_PWSonly_COV23.txt")
cat(paste0(g1, ";"))
sink(NULL)
        
tb23_only<-tb23[!(tb23$Gene_ID %in% overlaps), ]
g1<-unique(tb23_only)
sink("../Output/COV/COVscan_3pop/genes/GeneID_TBonly_COV23.txt")
cat(paste0(g1, ";"))
sink(NULL)

ss23_only<-ss23[!(ss23$Gene_ID %in% overlaps), ]
g1<-unique(ss23_only)
sink("../Output/COV/COVscan_3pop/genes/GeneID_SSonly_COV23.txt")
cat(paste0(g1, ";"))
sink(NULL)




#2. Between Time-Points within a population

times<-c("cov12","cov13","cov23")
pops<-c("PWS","TB")
common2<-list()
common_summary2<-data.frame(pop=rep(pops[1:2], each=4))
for (i in 1:length(pops)){
    plist<-GeneList[grep(pops[i], names(GeneList))]
    k=4*i-3
    #common genes between COV12 and COV13
    common_genes1<-intersect(plist[[1]]["Gene_name"], plist[[2]]["Gene_name"])
    common2[[k]]<-common_genes1
    names(common2)[[k]]<-paste0(pops[i],".", times[1],"_",times[2])
    common_summary2$Time[k]<-paste0(times[1],"_",times[2])
    common_summary2$no.of.genes[k]<-nrow(common_genes1) 
    
    c12<-plist[[1]]["Gene_name"]
    c13<-plist[[2]]["Gene_name"]
    c23<-plist[[3]]["Gene_name"]
    x<-list(COV12=c12$Gene_name,COV13=c13$Gene_name, COV23=c23$Gene_name)
    ggvenn(x, fill_color = cols[c(1,5,7)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle(pops[i])
    ggsave(paste0("../Output/COV/COVscan_3pop/Venn_",pops[i],".png"), width = 4, height=4, dpi=300)

    
    k=k+1
    #common genes between COV12 and COV23
    common_genes2<-intersect(plist[[1]]["Gene_name"], plist[[3]]["Gene_name"])
    common2[[k]]<-common_genes2
    names(common2)[[k]]<-paste0(pops[i],".", times[1],"_",times[3])
    common_summary2$Time[k]<-paste0(times[1],"_",times[3])
    common_summary2$no.of.genes[k]<-nrow(common_genes2) 
 
    k=k+1
    #common genes between COV13 and COV23
    common_genes3<-intersect(plist[[2]]["Gene_name"], plist[[3]]["Gene_name"])
    common2[[k]]<-common_genes3
    names(common2)[[k]]<-paste0(pops[i],".", times[2],"_",times[3])
    common_summary2$Time[k]<-paste0(times[2],"_",times[3])
    common_summary2$no.of.genes[k]<-nrow(common_genes3) 
 
    k=k+1
    #common genes among all time periods
    common_genes4<-intersect(plist[[1]]["Gene_name"], (intersect(plist[[2]]["Gene_name"], plist[[3]]["Gene_name"])))
    common2[[k]]<-common_genes4
    names(common2)[[k]]<-paste0(pops[i],".all")
    common_summary2$Time[k]<-"All"
    common_summary2$no.of.genes[k]<-nrow(common_genes4) 
}
write.csv(common_summary2, "../Output/COV/COVscan_3pop/Common_genes_betweenTimePoints.csv")


#Common gene names between time points

for (i in 1:2){
    CommonGenes<-data.frame()
    glist<-common2[grep(pops[i], names(common2))]
    for(j in 1:length(glist)){
        gids<-glist[[j]]
        df<-data.frame(Gene_name=gids)
        df2<-merge(df, Genes, by="Gene_name", all.x=T)
        write.csv(df2, paste0("../Output/COV/COVscan_3pop/Common_genes_", names(glist)[j],".csv"), row.names = F)
        df2$Time<-names(glist)[j]
        CommonGenes<-rbind(CommonGenes, df2)
    }
    write.csv(CommonGenes, paste0("../Output/COV/COVscan_3pop/Common_genes_",pops[i] ,".csv"), row.names = F)
}

4.3.0.1 Overlapping gene numbers

4.4 What are the genes overlapping across different time points between populations?

#1. between PWS and TB
pws.tb<-common_times[c(1,2,4)]

genes1213<-intersect(pws.tb[[1]]["Gene_name"], pws.tb[[2]]["Gene_name"])
genes1213<-merge(genes1213, Genes, by="Gene_name")
write.csv(genes1223, "../Output/COV/COVscan_3pop/Common_genes_PWS.TB.cov12-cov23.csv")
#           Gene_name   chr            Gene_ID
#1 ENSCHAG00000001687 chr13 ENSCHAG00000001687
#2             ndst2a chr13 ENSCHAG00000002649
#3             zswim8 chr13 ENSCHAG00000005956

## Common genes between population across time points (COV12 - COV13)
p1213<-pws.tb[[1]]["Gene_name"]
t1213<-pws.tb[[2]]["Gene_name"]
x<-list(PWS=p1213$Gene_name,TB=t1213$Gene_name)
ggvenn(x, fill_color = cols[c(2,1)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle("COV12-COV13 in PWS & TB")
ggsave(paste0("../Output/COV/COVscan_3pop/Venn_PWS_TB_COV12-COV13.png"), width = 3, height=3, dpi=300)
        
genes1223<-intersect(pws.tb[[1]]["Gene_name"], pws.tb[[3]]["Gene_name"])
genes1223<-merge(genes1223, Genes, by="Gene_name")
write.csv(genes1223, "../Output/COV/COVscan_3pop/Common_genes_PWS.TB.cov12-cov13.csv")
#           Gene_name   chr            Gene_ID
#1 ENSCHAG00000001687 chr13 ENSCHAG00000001687
#2 ENSCHAG00000022709 chr20 ENSCHAG00000022709
#3 ENSCHAG00000022815 chr20 ENSCHAG00000022815
#4             ndst2a chr13 ENSCHAG00000002649

p1223<-pws.tb[[1]]["Gene_name"]
t1223<-pws.tb[[3]]["Gene_name"]
x<-list(PWS=p1223$Gene_name,TB=t1223$Gene_name)
ggvenn(x, fill_color = cols[c(2,1)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle("COV12-COV23 in PWS & TB")
ggsave(paste0("../Output/COV/COVscan_3pop/Venn_PWS_TB_COV12-COV23.png"), width = 3, height=3, dpi=300)


genes1323<-intersect(pws.tb[[2]]["Gene_name"], pws.tb[[3]]["Gene_name"])
genes1323<-merge(genes1323, Genes, by="Gene_name")
write.csv(genes1323, "../Output/COV/COVscan_3pop/Common_genes_PWS.TB.cov13-cov23.csv")
#           Gene_name   chr            Gene_ID
#1 ENSCHAG00000001687 chr13 ENSCHAG00000001687
#2             ndst2a chr13 ENSCHAG00000002649

p1323<-pws.tb[[2]]["Gene_name"]
t1323<-pws.tb[[3]]["Gene_name"]
x<-list(PWS=p1323$Gene_name,TB=t1323$Gene_name)
ggvenn(x, fill_color = cols[c(2,1)], stroke_size = 0.5, set_name_size = 4,text_size=3)+ggtitle("COV13-COV23 in PWS & TB")
ggsave(paste0("../Output/COV/COVscan_3pop/Venn_PWS_TB_COV13-COV23.png"), width = 3, height=3, dpi=300)

5 Compare results from PH_MD7000 (all pops together) and 3Pops-NS0.5 (filter for 3 pops) VCF files

pws1<-read.csv("../Output/COV/COVscan_3pop/3pops_top1percent_outlier_regions.cov12_new.csv")
pws1<-pws1[pws1$pop=="PWS" & pws1$window=="100k",]
pws2<-read.csv("../Output/COV/3pops_top1percent_outlier_regions.cov12.csv")
pws2<-pws2[pws2$pop=="PWS"&pws2$window=="100k",]

pws1$vcf<-"3Pops"
pws2$vcf<-"PH"
pws<-rbind(pws1[,c("chrom","start","end","cov12","vcf")],pws2[,c("chrom","start","end","cov12","vcf")])
pws$chrom<-factor(pws$chrom, levels=paste0("chr", 1:26))
ggplot(data=pws,aes(x=start/1000000, y=cov12, color=vcf, fill=vcf))+
    facet_wrap(~chrom)+
    geom_point(position=position_dodge(width = 0.7), alpha=0.5)+xlab("Position (Mb)")+
    ggtitle("PWS COV12")
ggsave("../Output/COV/COVscan_3pop/PWS_PH.vs.3Pops_outlier_overlap_cov12.png", width = 10, height = 8, dpi=300)

pws1<-read.csv("../Output/COV/COVscan_3pop/3pops_top1percent_outlier_regions.cov23_new.csv")
pws1<-pws1[pws1$pop=="PWS" & pws1$window=="100k",]
pws2<-read.csv("../Output/COV/3pops_top1percent_outlier_regions.cov23.csv")
pws2<-pws2[pws2$pop=="PWS"&pws2$window=="100k",]

pws1$vcf<-"PH"
pws2$vcf<-"3Pops"
pws<-rbind(pws1[,c("chrom","start","end","cov23","vcf")],pws2[,c("chrom","start","end","cov23","vcf")])
pws$chrom<-factor(pws$chrom, levels=paste0("chr", 1:26))
ggplot(data=pws,aes(x=start/1000000, y=cov23, color=vcf, fill=vcf))+
    facet_wrap(~chrom)+
    geom_point(position=position_dodge(width = 0.7), alpha=0.5)+xlab("Position (Mb)")+
    ggtitle("PWS COV23")
ggsave("../Output/COV/COVscan_3pop/PWS_PH.vs.3Pops_outlier_overlap_cov23.png", width = 10, height = 8, dpi=300)

pws1<-read.csv("../Output/COV/COVscan_3pop/3pops_top1percent_outlier_regions.cov13_new.csv")
pws1<-pws1[pws1$pop=="PWS" & pws1$window=="100k",]
pws2<-read.csv("../Output/COV/3pops_top1percent_outlier_regions.cov13.csv")
pws2<-pws2[pws2$pop=="PWS"&pws2$window=="100k",]

pws1$vcf<-"PH"
pws2$vcf<-"3Pops"
pws<-rbind(pws1[,c("chrom","start","end","cov13","vcf")],pws2[,c("chrom","start","end","cov13","vcf")])
pws$chrom<-factor(pws$chrom, levels=paste0("chr", 1:26))
ggplot(data=pws,aes(x=start/1000000, y=cov13, color=vcf, fill=vcf))+
    facet_wrap(~chrom)+
    geom_point(position=position_dodge(width = 0.7), alpha=0.5)+xlab("Position (Mb)")+
    ggtitle("PWS COV13")
ggsave("../Output/COV/COVscan_3pop/PWS_PH.vs.3Pops_outlier_overlap_cov13.png", width = 10, height = 8, dpi=300)

6 Interpopulation comparison per time period

### Interpopulation comparisons
#decode the samples to create the right matrix
cv<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_3pops.csv", header = F)
labs<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_labels_3pops.csv" )
labs<-labs[,-1]
cvm<-data.frame(label=as.vector(t(labs)), cov=as.vector(t(cv)))

#rearrange based on comparions: covariance between populations within the same period
#PopYr Symbols
# PH 1 'PWS', 1991
# PH 2 'PWS', 1996
# PH 3 'PWS', 2006
# PH 4 'PWS', 2017
# PH 5 'SS',  1991
# PH 6 'SS',  1996
# PH 7 'SS',  2006
# PH 8 'SS',  2017
# PH 9 'TB',  1991
# PH 10'TB',  1996
# PH 11'TB',  2006
# PH 12'TB',  2017

Covs<-data.frame(pops=rep(c("PWS.vs.SS", "PWS.vs.TB",  "SS.vs.TB"), times=6),
                 period=c(rep("1991-1996", times=3),rep("1996-2006", times=3), rep("2006-2017", times=3)))

Covs$cov<-c(NA, cvm$cov[cvm$label=="cov(PH: 2-1, PH: 10-9)"],NA,
            cvm$cov[cvm$label=="cov(PH: 3-2, PH: 7-6)"],cvm$cov[cvm$label=="cov(PH: 3-2, PH: 11-10)"], 
            cvm$cov[cvm$label=="cov(PH: 7-6, PH: 11-10)"],
            cvm$cov[cvm$label=="cov(PH: 4-3, PH: 8-7)"],cvm$cov[cvm$label=="cov(PH: 4-3, PH: 12-11)"],cvm$cov[cvm$label=="cov(PH: 8-7, PH: 12-11)"])


#C.I.
cis<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_COV_Interpop_comparison_CIs.csv")
cis<-cis[,-1]
cim<-data.frame(label=as.vector(t(labs)), ci_l=as.vector(t(cis[1:11,])))
cim$ci_h<-as.vector(t(cis[12:22,]))

Covs$ci_l<-as.numeric(c(NA,cim$ci_l[cim$label=="cov(PH: 2-1, PH: 10-9)"],NA,
                      cim$ci_l[cim$label=="cov(PH: 3-2, PH: 7-6)"],cim$ci_l[cim$label=="cov(PH: 3-2, PH: 11-10)"], cim$ci_l[cim$label=="cov(PH: 7-6, PH: 11-10)"],
                      cim$ci_l[cim$label=="cov(PH: 4-3, PH: 8-7)"],cim$ci_l[cim$label=="cov(PH: 4-3, PH: 12-11)"], cim$ci_l[cim$label=="cov(PH: 8-7, PH: 12-11)"]))

Covs$ci_h<-as.numeric(c(NA, cim$ci_h[cim$label=="cov(PH: 2-1, PH: 10-9)"],NA,
                      cim$ci_h[cim$label=="cov(PH: 3-2, PH: 7-6)"],cim$ci_h[cim$label=="cov(PH: 3-2, PH: 11-10)"], cim$ci_h[cim$label=="cov(PH: 7-6, PH: 11-10)"],
                      cim$ci_h[cim$label=="cov(PH: 4-3, PH: 8-7)"],cim$ci_h[cim$label=="cov(PH: 4-3, PH: 12-11)"], cim$ci_h[cim$label=="cov(PH: 8-7, PH: 12-11)"]))

#Barplot
ggplot(Covs, aes(x=period, y=cov, fill=pops))+
    geom_bar(stat="identity",position=position_dodge(width = 0.7), width=0.8)+
    ylab("Covariance")+xlab('')+theme_classic()+
    geom_hline(yintercept = 0,color="gray70", size=0.3)+
    scale_fill_manual(values=cols[c(2,1,3)])+
    theme(legend.title = element_blank())+
    scale_y_continuous(labels = comma)+
    ylim(-0.0013, 0.002)+
    geom_errorbar(aes(ymin=ci_l, ymax=ci_h), width=.2, size=.2, position=position_dodge(width = 0.7))
ggsave("../Output/COV/Interpop_cov_comparison_3Pops_new.png",width = 4.7, height = 3, dpi=300)

#Point plot
ggplot(Covs, aes(x=period, y=cov, color=pops))+
    geom_point(position=position_dodge(width = 0.7), size=4)+
    ylab("Covariance")+xlab('')+theme_classic()+
    geom_hline(yintercept = 0,color="gray70", size=0.3)+
    scale_color_manual(values=cols[c(2,1,3)])+
    theme(legend.title = element_blank())+
    scale_y_continuous(labels = comma)+
    geom_errorbar(aes(ymin=ci_l, ymax=ci_h), width=.2, size=.2, position=position_dodge(width = 0.7))+
    ylim(-0.0013, 0.002)+
     geom_vline(xintercept = c(1.5,2.5), color="gray", size=0.3)
ggsave("../Output/COV/Interpop_cov_comparison_3Pops_new_PointPlot.png",width = 4.7, height = 3, dpi=300)

#line plot
Covs$time<-1
Covs$time[Covs$period=="1996-2006"]<-2
Covs$time[Covs$period=="2006-2017"]<-3
Covs<-Covs[order(Covs$time),]
ggplot(Covs, aes(x=time, y=cov, color=pops, group=pops))+
    geom_point(position=position_dodge(width = 0.7), size=4)+
    geom_path(position=position_dodge(width = 0.7))+
    ylab("Covariance")+xlab('')+theme_classic()+
    geom_hline(yintercept = 0,color="gray70", size=0.3)+
    scale_color_manual(values=cols[c(2,1,3)])+
    theme(legend.title = element_blank())+
    scale_y_continuous(labels = comma)+
    geom_errorbar(aes(ymin=ci_l, ymax=ci_h), width=.2, size=.2, position=position_dodge(width = 0.7))+
    ylim(-0.0013, 0.002)+
     geom_vline(xintercept = c(1.5,2.5), color="gray", size=0.3)+
    scale_x_continuous(breaks=c(1,2,3), labels = c("1991-1996","1996-2006","2006-2017"))
ggsave("../Output/COV/Interpop_cov_comparison_3Pops_new_LinePlot.png",width = 4.7, height = 3, dpi=300)

6.1 Longer time period

## Longer time-period
Covs2<-data.frame(pops=rep(c("PWS.vs.SS", "PWS.vs.TB",  "SS.vs.TB"), times=3),
                 period=c(rep("1991-2006", times=3),rep("1991-2017", times=3),rep("1996-2017", times=3)))

cv1<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_1991-2006.csv", header = F)
labs1<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_labels_1991-2006.csv" )
labs1<-labs1[,-1]
cvm1<-data.frame(label=as.vector(t(labs1)), cov=as.vector(t(cv1)))

cv2<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_1991-2017.csv", header = F)
labs2<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_labels_1991-2017.csv" )
labs2<-labs2[,-1]
cvm2<-data.frame(label=as.vector(t(labs2)), cov=as.vector(t(cv2)))

cv3<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_1996-2017.csv", header = F)
labs3<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_Covs_interPopulations_100k_labels_1996-2017.csv" )
labs3<-labs3[,-1]
cvm3<-data.frame(label=as.vector(t(labs3)), cov=as.vector(t(cv3)))

Covs2$cov<-c(NA, cvm1$cov[cvm1$label=="cov(PH: 2-1, PH: 4-3)"], NA,
             NA, cvm2$cov[cvm2$label=="cov(PH: 2-1, PH: 4-3)"], NA,
             cvm3$cov[cvm3$label=="cov(PH: 2-1, PH: 4-3)"], cvm3$cov[cvm3$label=="cov(PH: 2-1, PH: 6-5)"], cvm3$cov[cvm3$label=="cov(PH: 4-3, PH: 6-5)"])

#C.I.
cis1<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_COV_Interpop_comparison_CIs_1991-2006.csv")
cis1<-cis1[,-1]
cis2<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_COV_Interpop_comparison_CIs_1991-2017.csv")
cis2<-cis2[,-1]
cis3<-read.csv("~/Projects/Pacherring_Vincent/MD7000/GW_COV_Interpop_comparison_CIs_1996-2017.csv")
cis3<-cis3[,-1]

#cim<-data.frame(label=as.vector(t(labs)), ci_l=as.vector(t(cis1[1:4,])))
#cim$ci_h<-as.vector(t(cis[12:22,]))

Covs2$ci_l<-as.numeric(c(NA,cis1[1,3],NA,
                        NA,cis2[1,3],NA,
                      cis3[1,3],cis3[1,5],cis3[3,5]))

Covs2$ci_h<-as.numeric(c(NA,cis1[4,3],NA,
                        NA,cis2[4,3],NA,
                      cis3[6,3],cis3[6,5],cis3[8,5]))


ggplot(Covs2, aes(x=period, y=cov, fill=pops))+
    geom_bar(stat="identity",position=position_dodge(width = 0.7), width=0.8)+
    ylab("Covariance")+xlab('')+theme_classic()+
    geom_hline(yintercept = 0,color="gray70", size=0.3)+
    scale_fill_manual(values=cols[c(2,1,3)])+
    theme(legend.title = element_blank())+scale_y_continuous(labels = comma)+
    ylim(-0.0013, 0.002)+
    geom_errorbar(aes(ymin=ci_l, ymax=ci_h), width=.2, size=.2, position=position_dodge(width = 0.7))
ggsave("../Output/COV/Interpop_cov_comparison_3Pops_LonogerPeriod.png",width = 4.7, height = 3, dpi=300)

ggplot(Covs2, aes(x=period, y=cov, color=pops))+
    geom_point(position=position_dodge(width = 0.7), size=4)+
    ylab("Covariance")+xlab('')+theme_classic()+
    geom_hline(yintercept = 0,color="gray70", size=0.3)+
    scale_color_manual(values=cols[c(2,1,3)])+
    theme(legend.title = element_blank())+
    scale_y_continuous(labels = comma)+
     ylim(-0.0013, 0.002)+
    geom_errorbar(aes(ymin=ci_l, ymax=ci_h), width=.2, size=.2, position=position_dodge(width = 0.7))+
    geom_vline(xintercept = c(1.5,2.5), color="gray", size=0.3)
ggsave("../Output/COV/Interpop_cov_comparison_3PopsLonogerPeriod_PointPlot.png",width = 4.7, height = 3, dpi=300)

7 Focused freq analysis

7.1 rel gene (chr13: 23070000 - 23080000)

pops<-c("PWS91","PWS96","PWS07","PWS17")
yr<-c(1991,1996,2007,2017)
maf<-data.frame()
for (i in 1:4){
    af<-read.table(paste0("../Data/new_vcf/AF/",pops[i],".mafs"),sep="\t", header = T)
    af<-af[af$chromo=="chr13"&af$position>=23070000&af$position<=23080000,]
    af$year<-yr[i]
    maf<-rbind(maf,af)
}
#write.csv(maf,"../Output/COV/COVscan_3pop/AF_maf_chr13_23Mb.csv")

ggplot(maf, aes(x=year, y=knownEM, color=factor(position)))+
    geom_point()+
    geom_path()+ggtitle("MAF (ANGSD)")+
    ylab("maf")+
    theme(legend.title=element_blank())
ggsave("../Output/COV/COVscan/AF_ch13_23.07-23.08_angsd.png", width = 5, height=3, dpi=300)


AF<-data.frame()
for (i in 1:4){
    af<-read.table(paste0("../Data/new_vcf/AF/",pops[i],"_maf05_af.frq"),header = FALSE, skip=1, col.names = c("chr","pos","n_allele","n_sample","MajorAF","MAF"))
    af<-af[af$chr=="chr13"&af$pos>=23070000&af$pos<=23080000,]
    af$year<-yr[i]
    af$maf<-substr(af$MAF, 3,10)
    af$maf<-as.numeric(af$maf)
    af<-af[,c("chr","pos","maf","year")]
    AF<-rbind(AF,af)
}
ggplot(AF, aes(x=year, y=maf, color=factor(pos)))+
    geom_point()+
    geom_path()+ggtitle("MAF (vcftools)")+
    theme(legend.title=element_blank())
ggsave("../Output/COV/COVscan/AF_ch13_23.07-23.08.png", width = 5, height=3, dpi=300)


###TB
pops<-c("TB91","TB96","TB06","TB17","PWS91","PWS96","PWS07","PWS17","SS96","SS06","SS17")
yr<-c(1991,1996,2006,2017,1991,1996,2007,2017,1996,2006,2017)
maf<-data.frame()
for (i in 1:length(pops)){
    af<-read.table(paste0("../Data/new_vcf/AF/",pops[i],".mafs"),sep="\t", header = T)
    af<-af[af$chromo=="chr13"&af$position>=23070000&af$position<=23080000,]
    af$year<-yr[i]
    af$pop<-sub("\\d\\d","", pops[i])
    maf<-rbind(maf,af)
}
#write.csv(maf,"../Output/COV/COVscan_3pop/AF_maf_chr13_23Mb.csv")
ggplot(maf, aes(x=year, y=knownEM, color=pop))+
    facet_wrap(~factor(position))+
    geom_point()+
    geom_path()+ggtitle("Chr13 (rel gene)")+
    ylab("maf")+
    theme(legend.title=element_blank())
ggsave("../Output/COV/COVscan/AF_ch13_23.07-23.08_angsd.png", width = 5, height=3, dpi=300)